RESEARCH ARTICLE

Neuropeptide S reduces duodenal bicarbonate secretion and ethanol-induced increases in duodenal motility in rats Wan Salman Wan Saudi, Markus Sjo¨blom* Department of Neuroscience, Division of Physiology, Uppsala University, Uppsala, Sweden * [email protected]

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OPEN ACCESS Citation: Wan Saudi WS, Sjo¨blom M (2017) Neuropeptide S reduces duodenal bicarbonate secretion and ethanol-induced increases in duodenal motility in rats. PLoS ONE 12(4): e0175312. https://doi.org/10.1371/journal. pone.0175312 Editor: Yvette Tache, University of California Los Angeles, UNITED STATES Received: October 14, 2016 Accepted: March 23, 2017 Published: April 6, 2017 Copyright: © 2017 Wan Saudi, Sjo¨blom. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper.

Abstract Alcohol disrupts the intestinal mucosal barrier by inducing metabolic and functional changes in epithelial cells. Recently, we showed that neuropeptide S (NPS) decreases duodenal motility and increases mucosal paracellular permeability, suggesting a role of NPS in the pathogenesis of disorders and dysfunctions in the small intestine. The aim of the present study was to investigate the effects of NPS on ethanol- and HCl-induced changes of duodenal mucosal barrier function and motility. Rats were anaesthetized with thiobarbiturate, and a 30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ. The effects on duodenal bicarbonate secretion, the blood-to-lumen clearance of 51CrEDTA, motility and transepithelial net fluid flux were investigated. Intravenous (i.v.) administration of NPS significantly reduced duodenal mucosal bicarbonate secretion and stimulated mucosal transepithelial fluid absorption, mechanisms dependent on nitrergic signaling. NPS dose-dependently reduced ethanol-induced increases in duodenal motility. NPS (83 pmol
kg-1
min-1, i.v.) reduced the bicarbonate and fluid secretory response to luminal ethanol, whereas a 10-fold higher dose stimulated fluid secretion but did not influence bicarbonate secretion. In NPS-treated animals, duodenal perfusion of acid (pH 3) induced greater bicarbonate secretory rates than in controls. Pre-treating animals with Nω-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of NPS on bicarbonate secretion. In response to luminal acid, NPS-treated animals had significantly higher paracellular permeability compared to controls, an effect that was abolished by L-NAME. Our findings demonstrate that NPS reduces basal and ethanol-induced increases in duodenal motility. In addition, NPS increases luminal alkalinization and mucosal permeability in response to luminal acid via mechanisms that are dependent on nitric oxide signaling. The data support a role for NPS in neurohumoral regulation of duodenal mucosal barrier function and motility.

Funding: The current study was supported by Uppsala University (MS), the Ministry of Education of Malaysia (WSWS) and the Universiti Malaysia Sabah (WSWS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction

Competing interests: The authors have declared that no competing interests exist.

Long-term and excessive consumption of alcohol is associated with the etiology of liver disease [1]. However, moderate intake of alcohol has been shown to be beneficial by lowering the risk

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Ethanol- and acid-changes in duodenal barrier function

of coronary heart disease [2, 3]. After intake, alcohol is absorbed by the epithelium in the foregut, mainly in the duodenum but also to some minor extent in the stomach [4, 5]. Previous experiments from our laboratory showed that perfusing the duodenal lumen with 15% ethanol in saline induces potent increases in mucosal paracellular permeability [6]. Interestingly, the neuroendocrine hormone melatonin was shown to reduce the ethanol-induced increases in duodenal paracellular permeability partly via an enteric neural pathway in a dosedependent manner. Furthermore, melatonin was also shown to inhibit increases in duodenal motor activity in response to luminal ethanol [6, 7]. Although not fully described, it is plausible that the endogenous gastrointestinal neuroendocrine system may influence the magnitude of ethanol-induced changes to the regulation of tight junctional protein displacement and motility [6, 7]. Neuropeptide S (NPS), named after its serine residue, was recently shown to regulate duodenal motor activity and duodenal mucosal paracellular permeability [8]. In addition, previous studies have investigated the effects of NPS on regulating arousal, wakefulness and anxiety [9– 12]. In the gastrointestinal tract, NPS receptors (NPSR) are expressed by epithelial cells, enteroendocrine cells, leukocytes and smooth muscle cells [13–16], suggesting a broad role in regulating intestinal functions. Furthermore, a polymorphism in the NPSR gene was found to be highly expressed in mucosal epithelial tissues in asthma and inflammatory bowel disease (IBD) patients [14, 17], suggesting a role of NPS in inflammation. However, the role of NPS/NPSR in the regulation of duodenal barrier function in response to alcohol- or acid-induced post-prandial mucosal stress is not well characterized. The aim of the present study was to elucidate the effects of NPS on ethanol- and acidinduced changes of duodenal barrier function and motility in anesthetized rats in vivo. Duodenal bicarbonate secretion was determined by back titration of the perfusate, mucosal paracellular permeability was assessed by measuring the blood-to-lumen clearance of 51Cr-EDTA, and duodenal motor activity was determined by measuring changes in intraluminal pressure. Our results show that NPS significantly changed duodenal barrier function and motility in response to luminal ethanol and acid, suggesting a possible role of this peptide in the pathogenesis of intestinal functional and/or inflammatory reactions.

Materials and methods Chemicals and drugs 51

Chromium-labeled ethylenediaminetetraacetic acid (51Cr-EDTA) was purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Bovine albumin (A2153), sodium chloride (NaCl), hydrochloric acid (HCl), potassium chloride (KCl), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), the anesthetic 5-ethyl-5-(1’-methyl-propyl)-2-thiobarbiturate (Inactin1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Parecoxib (Dynastat1, Pfizer, NY, USA) were obtained from Apoteket AB (Uppsala, Sweden). Ethanol 95.5 vol-% (Etax A) was purchased from Solveco Chemicals AB (Ta¨by, Sweden). NPS was purchased from Bachem AG (Bubendorf, Switzerland).

Ethics statement All experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by Uppsala Ethics Committee for Experiments with Animals (Permit number: C309/10).

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Ethanol- and acid-changes in duodenal barrier function

Animals 60 male Sprague-Dawley rats weighing 300–350 g were obtained from Taconic, Ejby, Denmark. The animals were maintained under standardized temperature and light conditions (12:12-h light-dark cycle; temperature, 21–22˚C). The rats were acclimatized in the Animal Department for at least one week before experiments and were caged in groups of two or more with access to water and chow ad libitum (R36, Lantma¨nnen, Kimstad, Sweden). The animals were fasted but had free access to tap water for 16 hours (overnight) before the experiments. Experiments were started by anesthetizing the animal around 8 am with Inactin1 120 mg/kg body weight intraperitoneally. To minimize preoperative stress, anesthesia was performed within the Animal Department by the person who had previously handled the animals. Then, the rat was immediately transferred to the laboratory for surgical procedure.

Surgical procedure At the laboratory, the animals were tracheotomized with a tracheal tube to facilitate respiration, and the body temperature was maintained at 37–38˚C throughout the experiments using a heating pad controlled by a rectal thermistor probe. Two femoral arteries and femoral vein were catheterized with PE-50 polyethylene catheters (Becton, Dickinson & Co., Franklin Lakes, NJ, USA). For continuous recordings of the systemic arterial blood pressure, one of the arterial catheters containing 20 IU/ml heparin isotonic saline was connected to a transducer operating a PowerLab system (AD Instruments, Hastings, UK). The other arterial catheter was used for taking blood samples. The vein catheter was used for drug injections and for the infusion of saline and 51Cr-EDTA at a rate of 1.0 ml/h. A laparotomy was performed, and the common bile duct was catheterized with a PE-10 polyethylene tubing close to its entrance into the duodenum (2–3 mm) to prevent pancreatico-biliary juice from entering the duodenum. Soft silicone tubing (Silastic1, Dow Corning, 1 mm ID) was introduced into the mouth, pushed gently along the esophagus, guided through the stomach and pylorus, and secured by ligatures 2–5 mm distal to the pylorus. PE-320 tubing was inserted into duodenum approximately 2.5–3.5 cm distal to the pylorus and secured by ligatures. The proximal duodenal tubing was connected to a peristaltic pump (Gilson minipuls 3, Villiers, Le Bel, France), and the segment was continuously perfused with a 154 mM sodium chloride solution (saline) at a rate of ~0.4 ml/min. To complete the surgery, the abdominal cavity was closed with sutures, and the wound was covered with plastic foil. Parecoxib i.v. injection 10 mg/kg was given 30 min after completion of the surgery to reverse the surgery-induced paralysis of the intestine [18–20]. After surgery, ~60 min was allowed for the cardiovascular, respiratory, and intestinal function to stabilize before the experiments were commenced.

Measurement of luminal alkalinization The rate of luminal alkalinization was determined by back titration of the perfusate to pH 4.90 with 10 mM HCl under continuous gassing (100% N2) using pH-stat equipment (Autoburette ABU 901 and pH-stat controller PHM 290, Radiometer, Copenhagen, Denmark). During experiments perfusing the luminal segment with HCl pH 3, the collected perfusate was back titrated to a pH of 2.95 using 50 mM HCl. The pH electrode was routinely calibrated with standard buffers before the start of the titration. The amount of titrated HCl was considered equivalent to the duodenal mucosal HCO3ˉ secretion. The rates of luminal alkalinization are expressed as micromoles of base secreted per centimeter of intestine per hour (μmol
cmˉ1
hˉ1).

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Ethanol- and acid-changes in duodenal barrier function

Measurement of duodenal mucosal permeability After the completion of the surgery, 51Cr-EDTA was administered as an i.v. bolus of ~75 μCi followed by a continuous infusion at a rate of ~50 μCi per hour. The radioactive isotope was diluted in saline and infused at a rate of 1.0 ml/h. One hour was permitted for the tissue equilibration of the 51Cr-EDTA. Two blood samples (~0.2 ml each) were collected; the first was collected ten minutes before starting the experiment, and the second, after ending the experiment. The blood volume loss of the first blood sample was compensated by an injection of 0.2 ml of 7% bovine albumin solution. After centrifugation, 50 μl of the plasma was removed for measurements of radioactivity. The duodenal segment was perfused with saline at a rate of 0.4 ml/min, and the perfusate was collected at 10-min samples. The luminal perfusate and blood plasma were analyzed for 51 Cr-activity using a gamma counter (1282 Compugamma CS, Pharmacia, Uppsala, Sweden). A linear regression analysis of the plasma samples was made to obtain a corresponding plasma value for each perfusate sample. The clearance of 51Cr-EDTA from the blood to the lumen was calculated as described previously [21, 22] and expressed as (ml
minˉ1
100 gˉ1).

Measurement of duodenal wall contractions Measuring intraluminal pressure was used to assess the duodenal wall contractions. The inlet perfusion tubing was connected, via a T-tube to a pressure transducer, and intraluminal pressure was recorded on an IBM PC-compatible computer. The outlet tubing was positioned at the same level as the inlet tubing. An upward deflection of at least 2 mmHg above baseline was defined as a motor response. Changes in intraluminal pressure were recorded via a digitizer on a computer using PowerLab1 and the software Labchart71 (ADInstruments Ldt. Hastings, East Sussex, UK). The duodenal motility was assessed in intervals of 10 min by planimetry to measure the total area under the pressure curve (area under the curve; AUC) during the sample period. The values given are mean ± SEM of three 10-min intervals (unless stated otherwise).

Measurement of epithelial fluid flux The difference in the weight of the collection vials with and without perfusate was used to measure flow over a 10-min interval. The perfusate volumes were determined after correcting for density for each solution. The density of the isotonic saline was arbitrarily set to 1.0. The duodenum was perfused (~0.4 ml/min) with isotonic saline or other solutions, and the perfusate was collected every 10 min. The net fluid flux across the duodenal mucosa was determined by subtracting the perfusate volume per 10 min from the peristaltic pump volume per 10 min, and the result is expressed as ml fluid per gram of wet tissue weight per hour (ml
g-1
h-1). The peristaltic pump volume was determined from the mean of two 10-min samples taken immediately after the termination of each experiment.

Experimental protocol In all experiments, the rates of duodenal bicarbonate secretion (μmol
cm-1
h-1), mucosal paracellular permeability (ml
min-1
100 g-1), motility (area under the curve; AUC) and net fluid flux (ml
g-1
h-1) as well as systemic arterial blood pressure (mmHg) and body temperature (˚C) were monitored continuously and recorded at 10-min intervals. Control experiments were performed by measuring the parameters above during a 150min perfusion of isotonic saline (300 mOsm/kg) on the duodenal segment at a rate of ~0.4 ml/min.

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Ethanol- and acid-changes in duodenal barrier function

In the animals exposed to luminal ethanol 15%, hydrochloric acid pH 3 or a combination of ethanol 15% and hydrochloric acid pH 3, the experiment started with perfusion of the duodenum with saline for 30 min to collect baseline data. Then, the duodenum was perfused for 30 min with ethanol 15%, hydrochloric acid pH 3 or the combination of ethanol 15% and hydrochloric acid pH 3 solutions. For animals exposed to continuous i.v. NPS infusion, the experimental protocol was similar to the control experiment except that NPS was administered i.v. at 8, 83 and 833 pmol
kg-1
min-1 at 30, 70 and 110 min, respectively. Some groups of animals were pretreated 30 minutes before the experiment was started with an i.v. bolus of L-NAME (nitric oxide synthase, NOS inhibitor) 3 mg/kg followed by a 0.25 mg/h continuous i.v. infusion. In the latter group of animals, basal rates were recorded continuously 30 min prior administration of L-NAME. In the animals pretreated with i.v. NPS and exposed to luminal ethanol 15%, the experiment protocol was similar to that for animals exposed to luminal ethanol 15% except that an NPS infusion was administered i.v. at 83 or 833 pmol
kg-1
min-1 a few minutes before the experiment commenced. For animals pretreated with i.v. NPS and exposed to hydrochloric acid pH 3, the experiment protocol was exactly the same as for animals exposed to luminal hydrochloric acid pH 3 except that an NPS infusion was administered i.v. at 83 pmol
kg-1
min-1 starting a few minutes before the experiment started. Some groups of animals were pretreated 30 minutes before experiment was started with an i.v. bolus of L-NAME (nitric oxide synthase, NOS inhibitor) 3 mg/kg followed by a 0.25 mg/h continuous i.v. infusion. Immediately after finishing the experiment, the animal was euthanized with 1.0 ml bolus injection of 3 M KCl.

Statistical analysis Descriptive statistics are expressed as the mean ± SEM, with the number of experiments given in parentheses. The statistical significance of the data was tested by repeated measures analysis of variance (ANOVA). To test the differences within a group, a one-factor repeated measures ANOVA was used followed by a Tukey post-hoc test. Between groups, a two-way repeated measures ANOVA was used followed by a Bonferroni post-hoc test. Student’s paired t-test was used to test the differences within a group or between two groups. All statistical analyses were performed on an IBM-compatible computer using the Prism software package 6.05 (GraphPad Software Inc., San Diego, CA, USA). A P value less than 0.05 was considered significant.

Results In control animals (n = 9) where the duodenal segment was perfused with isotonic saline only, duodenal bicarbonate secretion, motility index and net fluid flux were stable at an average of 12.6±0.97 μmol
cm-1
h-1 (Fig 1A), 407±27 AUC/10 min (data not shown) and 0.58±0.15 ml
g-1
h-1 (Fig 1C), respectively, while duodenal mucosal paracellular permeability (blood-tolumen clearance of 51Cr-EDTA) decreased modestly in a linear fashion from the start to the end of experiment (from 0.36±0.04 to 0.28±0.04 ml
min-1
100 g-1; P